Abstract Human immunodeficiency virus (HIV) continues to cause a global epidemic with more than 36 million people currently living with HIV/AIDS. Following infection, HIV integrates its provirus into host genome and establishes highly stable latent infection, which has posed a major barrier to eradicate or cure the virus. In infected cells, integrated HIV transcription is subject to multiple layers of epigenetic regulation. Targeting host epigenetic machinery to durably repress integrated HIV and to induce an aviremic state free of ART is considered a novel approach to ?curing? HIV but has so far remained elusive. The Bromodomain (BD) and Extra-Terminal Domain protein (BET/BRD4) is an important epigenetic reader that interacts with cellular transcriptional machinery, such as super elongation complex (SEC), to control HIV gene expression. In fact, targeted modulation of BET/BRD4 by a pan BET inhibitor (JQ1) has been shown to activate latent HIV. In our preliminary studies, we have intriguingly identified a novel small molecule (named ZL0580) that selectively modulates human BRD4 but induces distinct functional and biochemical outcomes from JQ1. Unlike JQ1 which activates HIV, ZL0580 induces potent HIV suppression in multiple cell models, including J-Lat cells and HIV-infected human peripheral blood mononuclear cells (PBMCs) (both in vitro and ex vivo). Combinatorial treatment of HIV- infected PBMCs with ZL0580 and antiretroviral drugs (ART) indicates that ZL0580 accelerates HIV suppression during ART and delays viral rebound after ART removal. Mechanistically, our data indicate that ZL0580 induces HIV suppression by inhibiting multiple factors involved in Tat transactivation and transcription elongation. Therefore, our central hypothesis is that host BRD4 and its associated epigenetic machinery can be modulated to repress HIV, leading to enforced HIV latency and/or durable HIV silencing. In this application, we propose three related but independent specific aims to thoroughly investigate this novel compound in terms of its ex vivo repressive activity on latent HIV in human primary CD4 T cells (Aim 1), target validation (Aim 2) and mechanisms of action (Aim 3). We believe that this multidisciplinary project will be of high impact since it not only establishes a proof-of-concept for targeted modulation of host BRD4 to epigenetically suppress HIV, but also provides a promising lead chemical scaffold for this purpose. Our long-term goal is to develop these first-in-class molecules as novel tools and/or therapeutics for HIV epigenetic silencing.